Chronic myocardial ischemia leading to heart failure is a leading cause of morbidity and mortality in the United States. Experimental myocardial stem cell therapeutics have been performed largely in the acute ischemia model. The well established porcine hibernating myocardium model, which closely resembles the chronicity and stability of human coronary artery disease, will be used to develop and optimize therapeutic strategies based on combined stem cell and gene therapy. Bone marrow-derived porcine mesenchymal stem cells (MSCs) expanded in culture possess robust self-renewal and multilineage differentiation potentials, and are capable of producing many growth factors and cytokines. Although promising as regenerative medicine in aging and disease, MSCs await further analysis regarding the mechanisms governing their growth, differentiation, survival, tissue homing, and aging characteristics. Growth factor modulation of MSC multilineage potential, the influence of aging on the function of MSCs, and the use of allogeneic MSCs will be characterized. Central to these efforts is the use of recombinant adenovirus expressing genes involved in cytoprotection, angiogenesis, and MSC homing. The first part of the proposal relies on extensive cell culture characterizations of MSCs, building the foundation for the second part of the proposal that addresses the physiological effect of engineered MSCs. Aim 1 will determine the differential effects of multiple VEGF isoforms on the growth and multilineage potentials of porcine MSCs. Aim 2 will analyze the expression and regulation of MSC chemokine receptors involved in myocardial MSC homing. Aim 3 will characterize the influence of cellular and animal aging on MSC growth capability, cell survival capacity, multilineage potential, and chemotactic migratory potency. Aim 4 will optimize strategies for tracking and identifying the in vivo fate of implanted MSCs in the myocardium and evaluate the feasibility of using allogeneic and aged MSCs. Aim 5 will determine whether MSCs engineered for enhanced survival capacity, angiogenic potential, or homing potency can better improve flow and function in chronic hibernating myocardium. Long term, the translation between the MSC-based therapy in the porcine hibernating myocardium and regenerative medicine for humans with chronic coronary artery disease will lead to optimized MSC therapeutics that can be of clinical value in managing aging and curing disease.